Single-molecule quantification of 5-hydroxymethylcytosine for diagnosis of blood and colon cancers

Abstract

BackgroundThe DNA modification 5-hydroxymethylcytosine (5hmC) is now referred to as the sixth base of DNA with evidence of tissue-specific patterns and correlation with gene regulation and expression. This epigenetic mark was recently reported as a potential biomarker for multiple types of cancer, but its application in the clinic is limited by the utility of recent 5hmC quantification assays. We use a recently developed, ultra-sensitive, fluorescence-based single-molecule method for global quantification of 5hmC in genomic DNA. The high sensitivity of the method gives access to precise quantification of extremely low 5hmC levels common in many cancers.MethodsWe assessed 5hmC levels in DNA extracted from a set of colon and blood cancer samples and compared 5hmC levels with healthy controls, in a single-molecule approach.ResultsUsing our method, we observed a significantly reduced level of 5hmC in blood and colon cancers and could distinguish between colon tumor and colon tissue adjacent to the tumor based on the global levels of this molecular biomarker.ConclusionsSingle-molecule detection of 5hmC allows distinguishing between malignant and healthy tissue in clinically relevant and accessible tissue such as blood and colon. The presented method outperforms current commercially available quantification kits and may potentially be developed into a widely used, 5hmC quantification assay for research and clinical diagnostics. Furthermore, using this method, we confirm that 5hmC is a good molecular biomarker for diagnosing colon and various types of blood cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-017-0368-9) contains supplementary material, which is available to authorized users.

5‐Hydroxymethylcytosine as a clinical biomarker: Fluorescence‐based assay for high‐throughput epigenetic quantification in human tissues

“…5‐hmC was shown to have a functional role in gene expression regulation, and its global levels were found to be predominantly stable and highly tissue‐specific . A significant global reduction in 5‐hmC level was reported for various human cancers, such as melanoma, colorectal, pancreatic, breast, liver, lung, prostate, brain and blood cancers . This reduction suggests that 5‐hmC may serve as an important cancer biomarker, potentially enabling early‐stage detection.…”

“…Consequently, they often fail to detect small but significant differences in 5‐hmC content between samples. Resolving such differences is crucial for identification, staging and risk prediction of various medical conditions …”

Analytical epigenetics: single-molecule optical detection of DNA and histone modifications

“…Typically, fluorescently-labeled DNA is deposited or stretched on a microscope slide and imaged by a fluorescence microscope [26,27]. This enables precise quantification by single-molecule counting, while requiring only minute amounts of sample (~50 ng DNA) [28]. Thus, blood and colon cancer cells could be identified by their decreased 5hmC content despite the naturally low levels in these tissues [28] (Figure 3 (a)).…”

“…This enables precise quantification by single-molecule counting, while requiring only minute amounts of sample (~50 ng DNA) [28]. Thus, blood and colon cancer cells could be identified by their decreased 5hmC content despite the naturally low levels in these tissues [28] (Figure 3 (a)). Additionally, the method enables multiplexing, as demonstrated by assessing 5hmC positions and fluorescently-labeled DNA damage sites on the same DNA molecules [29].…”

“…(a) Enzymatic glycosylation of 5hmC is performed, then a fluorophore is attached. Fluorescence microscopy images on the right compare 5hmC levels in single DNA molecules from healthy colon and colorectal cancer tissue [28]. (b) Fluorescent labeling of non-methylated CpGs.…”